PROJECT SUMMARY The blood system is a developmental hierarchy maintained by hematopoietic stem cells (HSCs) at the apex. HSCs undergo self-renewal and multilineage differentiation into lineage-committed progenitors, giving rise to all mature blood cells. A major puzzle in developmental biology has been why hematopoietic ontogeny occurs in reverse order, with HSCs arising after lineage-committed progenitors initiate blood circulation. Similarly, it has been unclear why differentiation of pluripotent stem cells (PSCs) gives rise to hematopoietic progenitors, but fails to generate transplantable HSCs. Answering these questions would enable generation of HSCs from PSCs to overcome shortages of donor bone marrow for stem cell transplantation. We propose a novel paradigm that epigenetic factors restrict HSC fate by specifying closed chromatin conformation at promoters of HSC-specific genes, and the same mechanisms counteract directed differentiation of PSCs to HSC fate. We developed a functional multipotency assay in which PSC-derived myeloid lineage-committed progenitors are directed to T- and B-lymphoid differentiation. We will employ CRISPR-based loss-of- function screens to identify epigenetic factors which selectively repress multilineage potential. Using a panel of innovative approaches we will test the prediction that suppressing these factors can alter chromatin conformation at regulatory elements of HSC-specific genes, uncovering precocious stem cell potential in embryogenesis, and enabling specification of HSCs from PSCs. In addition, we will use single cell transcriptomics to probe the developmental transitions to hematopoietic cells. This molecular insight will further inform the selection of candidate factors for specifying long-term HSCs. This project is based on a novel paradigm which emphasizes the importance of negative regulation in fate determination, and involves highly innovative approaches to dissect its role in HSC formation. This project is poised to identify critical developmental regulators of stem cell fate and advance PSC- derived HSCs to the clinic.